This invention relates to the construction, isolation and identification of plasmids which contain DNA sequences directing the expression and secretion of specific classes of proteases and which facilely transform microorganisms to hyperproduce and secrete the specified proteases or other proteins.
Proteases are a group of enzymes which hydrolyze the peptide bonds of proteins. Proteases which are produced by bacteria can be classed in two general types. Those which are active at neutral pH and usually require a cofactor, such as zinc, to be active are called neutral proteases and can be inactivated by chelating agents such as ethylenediaminetetraacetic acid (EDTA) which remove the cofactor from the enzyme. Those which are active at high pH and cleave peptide bonds by a process analogous to alkaline hydrolysis are termed alkaline proteases. Alkaline proteases are also referred to as subtilisin and serine protease. Alkaline proteases can be inactivated by chemicals such as phenylmethylsulfonyl fluoride (PMSF) or by diisopropyl fluorophosphate (DFP). Bacterial proteases are available commercially from a number of suppliers (OPD Chemical Buyers Directory, A. P. Kavaler, ed, Schnell Publishing Co., Inc. New York, N.Y. 1982) and are used industrially to clarify beer, tan leather, tenderize meat, curdle milk and in the formulation of specialized detergents. Bacterial enzymes are extracted from cultures of specific strains of microorganisms, many of which are the result of careful selction and of artificial mutation (e.g. UV irradiation). Limitations on the production of enzymes include the rate at which the microorganism produces the enzyme, the degree to which the microorganism excretes the enzyme produced, and the stability of the microorganism in culture.
In recent years, processes have been developed for inserting into a bacterial organism a gene or genes from another organism so that the bacterium will produce "foreign" proteins. The technique for introducing the DNA which directs ("codes for") the production of an enzyme involves the cleavage of DNA from the source cell using one of a number of "restriction endonucleases", attaching ("ligating") the cleaved DNA to DNA from a plasmid to form a "vector," and introducing the vector into the host under conditions which make the "transformation" successful.
The organisms of the genus Bacillus are aerobes and have been widely used in the fermentation industry because they are non-pathogenic and, in the case of protein production, because they have the ability to secrete proteins. Most research into recombinant DNA has been performed using E. coli as the host. E. coli is generally a non-secreting microorganism well-developed for the cloning of recombinant plasmids but not well suited for the commercial production of proteins. Bacillus species, particularly B. subtilis, cannot be transformed using monomer plasmid or conventional techniques developed for E. coli. (Canosi et al. Mol. gen. Genet., 166:259 [1978], Keggins et al., Proc. Nat. Acad. Sci. U.S., 75:1423 [1978] and Michel et al., Gene, 12:147 [1978]). A number of alternative strategies have been developed for the transformation of B. subtilis, including the use of plasmid multimers (Canosi), the use of polyethylene glycol to induce DNA uptake in protoplasts (Chang S. and Cohen S.N., Mol. gen. Genet., 168:111 [1979]) and the use of the marker rescue technique (Gryczan et al., Mol. gen. Genet., 177:459 [1980]). A structural gene for alpha-amylase, including the associated control regions, from B. amyloliquefaciens has been shotgun cloned into B. subtilis. The alpha-amylase was expressed in B. subtilis at a rate five times that produced by the source B. amyloliquefaciens (Palva I. Gene, 19:81-87 [1982]).
A Bacillus transformant, e.g. a B. subtilis transformant, expressing large amounts of proteases would have particular commercial importance. Moreover, an efficient expression vector for transforming B. subtilis which includes a promoter, operator and ribosome binding site, as well as the structural gene for a specific enzyme such as a protease would be useful not only because it could be used to transform B. subtilis to produce the enzyme, but also because it could provide an efficient promoter and regulatory region which could be used for the expression and secretion of heterologous proteins in B. subtilis.